Investigation Of The Biological Activity Of Proline Analogues And Zn²⁺Tetraazamacrocycle Complex And Their Regulation On HIF-1? Signaling Pathway

Hypoxia inducible factor(HIF)plays a pivotal role in regulator of oxygen homeostasis.HIF mediates the transcription of a series of downstream target genes that participate in angiogenesis,glucose metabolism and cell proliferation/survival etc.HIF acts an important role in hypoxia ischemia disease and cancer.Hence,activating and inhibiting the levels of HIF-la are potential targets for treating HIF-related diseases.In this thesis,effects of proline analogues(PA1 and PA2)on activity of prolyl hydroxylases(PHD3)and regulation of HIF signal transduction pathway were studied.Zn2+tetraazamacrocycle complex(L-Zn)was used as an anticancer agent to investigate its antitumor activity in vivo and in vitro?inhibition of HIF-la signal transduction pathway and apoptosis mechanism.This dissertation mainly includes three parts as follows:(1)Soluble and active PHD3 was expressed in the E.coli and purified by Ni-NTA agarose column.A fluorescence-based assay based on derivatisation of o-phenylenediamine with unreacted 20G was applied to study the enzyme kinetics.The inhibition of PHD3 activity by proline analogues PA1 and PA2 was studied.PAl and PA2,which were not ferrous chelating agents,could inhibit PHD3 activity in a different manner.PA1 was substrate competitive inhibitor while PA2 was substrate non-competitive inhibitor with the apparent IC50 values were 1.53 and 3.17 ?M,respectively.PA1 showed good inhibition potency of PHD3 activity.RT-PCR?Western-blot and ELISA were used to study the regulation of HIF-VEGF signaling pathway by PA1 in NCI-H446 cells.The results showed that PA1 significantly raised HIF-la protein levels and promoted the expression of VEGF mRNA and protein.Moreover,2-DE coupled with MALDI-TOF/TOF-MS technologies were used to detect the differentially expressed proteins between control and PAl-treated groups.Nine proteins were identified to significantly regulated by PA1,among which,Pyruvate kinase isozymes M1/M2(PKM)and alpha-enolase 1(ENO1)are key modulators of glycolysis and are directly regulated by HIF-1?.PA1 could be used as a potential inhibitor of PHD3,which provided innovative evidence for the treatment of HIF-related diseases.(2)MTT assay was used to test cytotoxicity of L-Zn.The results showed that L-Zn exerted greater cytotoxicity than cisplatin in several human cancer cell lines and lower cytotoxicity in LO2 cells.RT-PCR and Western-blot assays were detected the variations of HIF-1? mRNA and protein expression levels which were significantly decreased in a dose-dependent manner by L-Zn treated HepG2 cells for 24 h.L-Zn also lead to a significant reduction of HIF-1? target genes GLUT 1 and VEGF mRNA and protein expression levels,which inhibited angiogenesis and glucose metabolism to inhibiting cancer cell proliferation.ICR mice subcutaneous H22 hepatoma cell models also used to assess antitumor activity of L-Zn in vivo.The antitumor activity increased significantly with increasing concentration of L-Zn(from 21.7%to 40.9%).L-Zn showed well antitumor activity in the cancer cells and tumor model mice tests and can significantly decrease the expression of HIF-la and inhibit its downstream target gene expression(3)Nuclear staining and flow cytometry assays were used to detect the effect of L-Zn on HepG2 cells apoptosis.The results revealed that nuclei condensed and fragmented,the percentage of apoptotic cells increased to 41.7%,the cell cycle was arrested at G2/M phase and sub-G1 phase appeared and increased.L-Zn could be effective to induce apoptosis of HepG2 cells.L-Zn promoted ROS and Ca2+productions and decreased the levels of mitochondrial membrane potential(??m).2-DE coupled with MALDI-TOF/TOF MS technologies were used to detect the differentially expressed proteins between control and L-Zn-treated groups.Eighteen proteins were identified and their functions were involved in cell metabolism,gene expression and cell apoptosis.Among the eighteen proteins,GRP78 and Erp29 are related to ER stress,RCN1 is a calcium binding protein which can regulate the calcium level,ANXA5 is participated in mitochondrial apoptosis pathway.Western-blot assay was used to detect the expression of proteins that related to apoptosis.The mechanism of L-Zn induced cell apoptosis is complex.L-Zn promoted ROS which could directly damage DNA and induce cell apoptosis.L-Zn also downregulated the expression of GRP78,Erp29 and of RCN1 to destroy endoplasmic reticulum structure,led to raise the level of intracellular Ca2+to transmit apoptosis signals to the mitochondria.L-Zn up-regulated ANXA5 and Bax,down-regulated Bcl-2,induced AIF and Endo G transfer from mitochondria to nuclei and led to HepG2 cell apoptosis in a caspases-independent apoptosis pathway.